Many agrochemicals and pharmaceuticals of the general formula X--CHR--COOH are currently marketed as racemic or diastereomer mixtures. In many cases the physiological effect derives from only one enantiomer/diastereomer where the other enantiomer/diastereomer is inactive or even harmful. Methods for synthesizing enantiomers are becoming increasingly important tools for the production of chemicals of enantiomer purity. To date, however, no recombinant, stereospecific NHase has been described capable of catalyzing the hydrolysis of certain racemic nitrites to the corresponding R- or S-amides.
Methods for the selective preparation of stereo-specific amides from nitrites are known and incorporate microorganisms possessing nitrile hydratase activity (NHase). These NHases catalyze the addition of one molecule of water to the nitrile, resulting in the formation of the amide free product according to Reaction 1:
Reaction 1 R--CN+H.sub.2 O.fwdarw.RCONH.sub.2
Similarly, methods for the stereospecific production of carboxylic acids are known and incorporate microorganisms possessing an amidase (Am) activity. In general amidases convert the amide product of Reaction 1 to the acid free product plus ammonia according to Reaction 2:
Reaction 2 RCONH.sub.2 .fwdarw.RCOOH+NH.sub.3
A wide variety of bacterial genera are known to possess a diverse spectrum of nitrile hydratase and amidase activities including Rhodococcus, Pseudomonas, Alcaligenes, Arthrobacter, Bacillus, Bacteridium, Brevibacterium, Corynebacterium, and Micrococcus. For example, nitrile hydratase enzymes have been isolated from Pseudomonas chlororaphis, B23 [Nishiyama, M. J., Bacteriol., 173:2465-2472 (1991)] Rhodococcus rhodochrous J1 [Kobayashi, M., Biochem. Biophys. Acta, 1129:23-33 (1991)] Brevibacterium sp. 312(Mayaux et al., J. Bacteriol., 172:6764-6773 (1990)), and Rhodococcus sp. N-774 [Ikehata, O., Nishiyama, M., Horinouchi, S., Beppu, T., Eur. J. Biochem., 181: 563-570(1989)). No disclosure of any stereoselective activity is made for any of these enzymes. Only two disclosures have been made for stereoselective nitrile hydratase activity in native bacterial strains. The Applicants have disclosed a stereospecific nitrile hydratase from P. putida NRRL-18668 [WO 92/05275 (1990)].
Wildtype microorganisms known to possess nitrile hydratase activity have been used to convert nitrites to amides and carboxylic acids. For example, EPA 326,482 discloses the stereospecific preparation of aryl-2-alkanoic acids such as 2-(4-chlorophenyl)-3-methylbutyric acid by microbial hydrolysis of the corresponding racemic amide using members of Brevibacterium and Corynebacterium. Similarly, U.S. Pat. No. 4,366,250 teaches the use of Bacillus, Bacteridium, Micrococcus and Brevibacterium in a method for the preparation of L-amino acids from the corresponding racemic amino nitrites. Finally, WO 92/05275 teaches a biologically-catalyzed method for converting a racemic alkyl nitrile to the corresponding R- or S-alkanoic acid through an intermediate amide using members of the bacterial genera Pseudomonas spp. (e.g., putida, aureofaciens, Moraxella spp.) and Serratia (e.g., Serratia liquefaciens)
In addition to the use of wildtype organisms, recombinant organisms containing heterologous genes for the expression of nitrile hydratase are also known for the conversion of nitrites. For example, Cerebelaud et al., (WO 9504828) teach the isolation and expression in E. coli of nitrile hydratase genes isolated from C. testosteroni. The transformed hosts effectively convert nitrites to amides where the nitrile substrate consists of one nitrile and one carboxylate group. However, WO 9504828 does not teach a stereospecific conversion of nitrites.
Similarly, Beppu et al., (EP 5024576) disclose plasmids carrying both nitrile hydratase and amidase genes from Rhodococcus capable of transforming E. coli where the transformed host is then able to use isobutyronitrile and isobutyroamide as enzymatic substrates. However, EP 5024576 does not teach a stereospecific conversion of nitrites or amides.
As with nitrile hydratases, microorganisms possessing amidase activity have been used to convert amides to carboxylic acids. In U.S. Ser. No. 08/403,911, Applicants disclose a method for converting an (S)-amide, or stereospecifically converting a mixture of (R)- and (S)-amides to the corresponding enantiomeric (S)-carboxylic acid by contacting said amide with Pseudomonas chlororaphis B23 in a solvent. This method uses a wildtype microorganism and does not anticipate a recombinant catalyst or heterologous gene expression. Blakey et al., FEMS Microbiology Letters, 129:57-62 (1995) disclose a Rhodococcus sp. having activity against a broad range of nitrites and dinitriles and able to catalyze regio-specific and stereo-specific nitrile biotransformations.
Genes encoding amidase activity-have been cloned, sequenced, and expressed in recombinant organisms. For example, Azza et al., (FEMS Microbiol. Lett. 122, 129, (1994)) disclose the cloning and over-expression in E. coli of an amidase gene from Brevibacterium sp. R312 under the control of the native promoter. Similarly, Kobayashi et al., (Eur. J. Biochem., 217, 327, (1993)) teach the cloning of both a nitrile hydratase and amidase gene from R. rhodococcus J1 and their co-expression in E. coli.
What is needed and inventive over the prior art is a method for the stereospecific conversion of racemic alkyl nitriles to the corresponding R- or S-alkanoic acids using a recombinant organism.